Introduction
The general idea of electrically insulating a patch of membrane and studying the ion channels in that patch under voltage-clamp conditions is outlined in Neher, Sakmann, and Steinback (1978) “The Extracellular Patch Clamp, A Method For Resolving Currents Through Individual Open Channels In Biological Membranes”, Pflüger Arch. 375;219–278. It was found that, by pressing a pipette containing acetylcholine (ACh) against the surface of a muscle cell membrane, one could see discrete jumps in electrical current that were attributable to the opening and closing of ACh-activated ion channels. However, the researchers were limited in their work by the fact that the resistance of the seal between the glass of the pipette and the membrane (10–50 MΩ) was very small relative to the resistance of the channel (10 GΩ). The electrical noise resulting from such a seal is inversely related to the resistance and, consequently, was large enough to obscure the currents flowing through ion channels, the conductance of which are smaller than that of the ACh channel. It also prohibited the clamping of the voltage in the pipette to values different from that of the bath due to the resulting large currents through the seal.
It was then discovered that by fire polishing the glass pipettes and by applying suction to the interior of the pipette a seal of very high resistance (1 to 100 GΩ) could be obtained with the surface of the cell, thereby reducing the noise by an order of magnitude to levels at which most channels of biological interest can be studied and greatly extended the voltage range over which these studies could be made. This improved seal has been termed a ‘gigaseal’, and the pipette has been termed a ‘patch pipette’. A more detailed description of the gigaseal may be found in O. P. Hamill, A. Marty, E. Neher, B. Sakmann & F. J. Sigworth (1981) “Improved patch-clamp techniques for high resolution current recordings from cells and cell-free membrane patches.” Pflügers Arch. 391, 85–100. For their work in developing the patch clamp technique, Neher and Sakmann were awarded the 1991 Nobel Prize in Physiology and Medicine.
Ion channels are transmembrane proteins which catalyse transport of inorganic ions across cell membranes. The ion channels participate in processes as diverse as generating and timing action potentials, synaptic transmission, secretion of hormones, contraction of muscles, etc. Many pharmacological agents exert their specific effects via modulation of ion channels. Examples include antiepileptic compounds such as phenytoin and lamotrigine, which block voltage-dependent Na+-channels in the brain, antihypertensive drugs such as nifedipine and diltiazem, which block voltage dependent Ca2+-channels in smooth muscle cells, and stimulators of insulin release such as glibenclamide and tolbutamide, which block an ATP-regulated K+-channel in the pancreas. In addition to chemically-induced modulation of ion-channel activity, the patch clamp technique has enabled scientists to perform manipulations with voltage-dependent channels. These techniques include adjusting the polarity of the electrode in the patch pipette and altering the saline composition to moderate the free ion levels in the bath solution.
The Patch Clamp Technique
The patch clamp technique represents a major development in biology and medicine, since it enables measurement of ion flow through single ion channel proteins, and also enables the study of a single ion channel activity in response to drug exposure. Briefly, in standard patch clamping, a thin (approx. 0.5–2 μm in diameter) glass pipette is used. The tip of this patch pipette is pressed against the surface of the cell membrane. The pipette tip seals tightly to the cell membrane and isolates a small population of ion channel proteins in the tiny patch of membrane limited by the pipette orifice. The activity of these channels can be measured individually (‘single channel recording’) or, alternatively, the patch can be ruptured, allowing measurements of the channel activity of the entire cell membrane (‘whole-cell configuration’). High-conductance access to the cell interior for performing whole-cell measurements can be obtained by rupturing the membrane by applying negative pressure in the pipette.
The Gigaseal
As discussed above, an important requirement for patch clamp measurements of single-channel currents is the establishment of a high-resistance seal between the cell membrane and the glass micropipette tip, in order to restrict ions from moving in the space between the two surfaces. Typically, resistances in excess of 1 GΩ are required, hence the physical contact zone is referred to as a ‘gigaseal’.
Formation of a gigaseal requires that the cell membrane and the pipette glass are brought into close proximity to each other. Thus, while the distance between adjacent cells in tissues or between cultured cells and their substrates generally is in the order of 20–40 nm (Neher, 2001), the distance between the cell membrane and the pipette glass in the gigaseal is predicted to be in the Angstrom (i.e. 10–10 m) range. The physico-chemical nature of the gigaseal is not known. However, gigaseals may be formed between cell membranes and a wide variety of glass types including quartz, aluminosilicate, and borosilicate (Rae and Levis, 1992), indicating that the specific chemical composition of the glass is not crucial.
Cell Membrane Structure
Cell membranes are composed of a phospholipid bilayer with intercalated glycoproteins, the latter serving a multitude of functions including acting as receptors for various agents. These membrane-spanning glycoproteins typically comprise peptide- and glyco-moieties which extend out from the membrane into the extracellular space, forming a so-called ‘glycocalyx’ layer around the phospholipid bilayer which reaches a height of 20 to 50 nm and creates an electrolyte-filled compartment adjacent to the phospholipid bilayer (see FIG. 1). Thus, the glycocalyx forms a hydrophilic and negatively charged domain constituting the interspace between the cell and its aqueous environment.
Cytoskeleton and Glycocalyx
Immediately underneath the cell membrane is located the cytoskeleton, a meshwork of actin filaments, spectrin, anchyrin, and a multitude of other large structural molecules. One important role of the cytoskeleton is to anchor certain integral membrane proteins and glycoproteins to fixed positions within the membrane. However, it is believed that intercalated membrane glycoproteins are free, within certain limits (lipid micro domains or ‘rafts’; for a review see Simons and Toomre, 2000), to move laterally in the phospholipid bilayer. Indeed, such an arrangement has been described as being ‘like protein icebergs in an ocean of lipids’.
Effect of Glycocalyx on Gigaseal Formation
In conventional patch clamp methods, the initial point of contact between the glass pipette tip (which has a wall thickness of approximately 100 nm) and the cell involves the glycocalyx. An estimation of the electrical resistance, represented by the 150 mM electrolyte contained in the inter-space defined between the glass surface and the lipid membrane, by the height of the glycocalyx (e.g. 20 to 40 mn) results in 20–60MΩ. This estimation is in agreement with experimental observations on smooth surface quartz coated chips of the TEOS (Triethyloxysilane) type, which routinely yield resistances in the order of 40 MΩ (or only 4% of a GΩ). In this estimation, it is assumed that the electrolyte is present between the lipid membrane and a glass surface approximately of cylindrical shape with diameter about 1 μm and length about 3–10 μm. Subsequent gentle suction (<20 hPa) applied to the pipette further increases the resistance, ideally leading to a gigaseal. Gigaseal formation may take place rapidly on a time scale of 0.1 to 10 s, or it may be a prolonged process completed only after several successive rounds of increased suction pressure. The time course of the gigaseal formation, reflects the exclusion of glycoproteins from the area of physical (membrane/pipette) contact by lateral displacement in the ‘liquid-crystal’ phospholipid bilayer. In other words, the elements of the glycocalyx, i.e. glycoproteins, are squeezed out of the area of contact due to the negative hydrostatic pressure applied to the pipette which forces the phospholipid bilayer (the hydrophilic polar heads of the phospholipids) against the glass surface (hyydrophilic silanol groups).
However, sometimes the process of resistance increase proceeds only up to formation of a quasi gigaseal (0.5 to 1 GΩ). Empirically, application of a large (50–70 mV; Penner, 1995) negative electrical potential to the pipette at this point may lead to the final resistance increase terminating with the gigaseal. In terms of the glycocalyx, the latter observation may be explained by negatively charged domains of glycoproteins being displaced laterally driven by the applied negative pipette potential. The strength of the electrical field (E) acting on the glycoproteins, i.e. the electrical field from pipette lumen to the surrounding bath is considerable:
  E  =            x      V        =                            70          ⁢                                          ⁢          mV                          100          ⁢                                          ⁢          nm                    =              700.000        ⁢                                  ⁢                  V/m                    assuming a pipette tip wall thickness (x) of 100 nm and an applied pipette potential (V) of −70 mV.Conventional Pipettes Versus Planar Substrates
Recent developments in patch clamp methodology have seen the introduction of planar substrates (e.g. a silicon chip) in place of conventional glass micropipettes (for example, see WO 01/25769 and Mayer, 2000).
Attempts to form gigaseals between planar silicon-based chips and living cells have proven problematic (for example, see Mayer, 2000). However, success has been achieved in obtaining gigaseals between artificial phospholipid vesicles which contain no exterior glycocalyx. This finding indicates a critical importance of the glycocalyx in the gigaseal formation process.
Hence, there is a need for improved planar substrates suitable for use in patch clamp studies of cell membrane electrophysiology which permit the formation of a gigaseal with cell membranes comprising a glycocalyx.